INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 40: 1477-1485, 2017
Fibroblast growth factor 21 protects rat cardiomyocytes from endoplasmic reticulum stress by promoting the fibroblast growth factor receptor 1-extracellular signal‑regulated kinase 1/2 signaling pathway Pingping Liang1*, Lin Zhong1*, Lei Gong2, Jiahui Wang3, Yujie Zhu1, Weifeng Liu1 and Jun Yang1 1
Department of Cardiology, 2Biochip Laboratory, and 3Central Laboratory, Yantai Yuhuangding Hospital, Affiliated Hospital of Medical College Qingdao University, Yantai, Shandong 264000, P.R. China Received February 25, 2017; Accepted September 8, 2017 DOI: 10.3892/ijmm.2017.3140
Abstract. Fibroblast growth factor 21 (FGF21), as an endocrine factor, is secreted into circulation by injured cardiomyocytes. Endoplasmic reticulum (ER) stress-induced apoptosis has been proposed as an important pathophysiological mechanism for cardiomyocyte injury. However, whether the enhanced expression of FGF21 in cardiomyocytes is linked to ER stress, and the effect and underlying mechanism of FGF21 on ER stress-induced cardiomyocyte apoptosis remain unclear. In the present study, it was demonstrated that mild ER stress resulted in upregulated expression levels of FGF21 and its main receptors, as a response to cell compensation, at the induction of ≤5 µM tunicamycin (TM). However, excessive ER stress (TM ≥10 µM) activated the ER stress-mediated apoptosis signaling pathways, including PKR-like ER kinase (PERK)-eukaryotic translational initiation factor 2α (eIF2α)-activating transcrip-
tion factor 4 (ATF4)-CCAAT/-enhancer-binding protein homologous protein (CHOP) and inositol-requiring kinase 1α (IRE1α)-c-Jun N-terminal kinases (JNK), as well as inhibited the expression of FGF21 and its primary receptors. In addition, FGF21 overexpression provided protection against ER stress-induced cardiomyocyte injury, as evidenced by increased cell viability and reduced apoptosis. These changes were associated with the inhibition of ER stress-mediated apoptosis signaling pathways, as well as increased phosphorylation of FGFR1 and ERK1/2. However, the protective effects of overexpressed FGF21 were abolished following treatment with FGFR1 and ERK1/2 inhibitors. Thus, mild ER stress may induce the expression of FGF21 and its primary receptors in cardiomyocytes. FGF21 inhibits ER stress-induced cardiomyocyte injury as least in part via the FGFR1-ERK1/2 signaling pathway. Introduction
Correspondence to: Dr Jun Yang, Department of Cardiology, Yantai Yuhuangding Hospital, Affiliated Hospital of Medical College Qingdao University, 20 Yuhuangding East Road, Yantai, Shandong 264000, P.R. China E-mail: [email protected] *
Contributed equally
Abbreviations: FGF21, fibroblast growth factor 21; ER, endo plasmic reticulum; UPR, unfolded protein response; PERK, PKR-like ER kinase; IRE1, inositol-requiring kinase 1α; ATF6, activating transcription factor 6; eIF2α, eukaryotic translational initiation factor 2α; ATF4, activating transcription factor 4; XBP1, X-box binding protein 1; JNK, c-Jun N-terminal kinase; CHOP, CCAAT/-enhancer-binding protein homologous protein; c-caspase-3, cleaved caspase-3; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2 associated X, apoptosis regulator; ERK, extracellular signalregulated kinases; TM, tunicamycin Key words: fibroblast growth factor 21, apoptosis, endoplasmic reticulum stress, cardiomyocytes, fibroblast growth factor receptor 1, extracellular signal-regulated kinase 1/2
Fibroblast growth factor 21 (FGF21) is a unique member of the FGF superfamily, which is predominantly expressed in the liver and exerts pleiotropic effects on metabolic processes (1). FGF21 may activate FGF receptors (FGFRs), including FGFR1-4, which all contain an intracellular tyrosine kinase domain, and FGFR1 is the predominant FGFR isotype in the heart and vessels (2). β-Klotho, as a co-receptor, is necessary for FGF21 to bind with particular FGFRs and effectively activate the FGF21 signaling pathway (3). Furthermore, among the various isoforms of FGFRs, FGFR1 has been suggested as the primary form for FGF21 in cardiomyocytes and the function of FGF21 was mediated primarily by binding to FGFR1 in a β -Klotho-dependent manner (2,4). A previous study identified that FGF21 is upregulated and released into circulation by the liver tissue in response to myocardial ischemia (5). Serum levels of FGF21 are also elevated in patients with coronary artery disease, acute myocardial infarction and heart failure (6-8). Additionally, FGF21 may decrease ischemia-reperfusion injury of cardiomyocytes and improve the antioxidant capacity of endothelial cells (9,10), indicating that FGF21 is a critical protective factor for the cardiovascular system.
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LIANG et al: FGF21 PROTECTS RAT CARDIOMYOCYTES FROM ENDOPLAMIC RETICULUM STRESS
Accumulation of unfolded or misfolded proteins at the reticulum transmembrane causes endoplasmic reticulum (ER) stress, activating an adaptive response termed the unfolded protein response (UPR) (11). The UPR involves the PKR-like ER kinase (PERK), inositol-requiring kinase 1α (IRE1α) and activating transcription factor 6 (ATF6) (12), which all regulate the transcription of a variety of associated genes encoding ER chaperone proteins to clear the unfolded protein aggregation at the initial stage of ER stress. When ER stress is prolonged and/or excessive, the UPR fails to control the level of unfolded or misfolded proteins in the ER, subsequently leading to the activation of apoptotic pathways (11-13). Proteins downstream of the PERK and IRE1 signaling pathways have been identified as having pro-apoptotic roles. PERK phosphorylates eIF2α to induce the translation of ATF4. ATF4 modulates a wide range of genes involved in the adaptation to ER stress and induces CCAAT/-enhancer-binding protein homologous protein (CHOP) to initiate the apoptosis signaling pathway (14,15). IRE1α mediates the accumulation of misfolded proteins, and induces downstream molecular X-box binding protein 1 (XBP1) and c-Jun N-terminal kinase (JNK). However, IRE1α-dependent apoptotic signaling occurs via phosphorylating JNK (16), which induces cell death via the induction of various proteins from the B-cell lymphoma-2 (Bcl-2) family and the activation of cysteinyl aspartate specific proteinase (caspases) (17,18). Evidence indicates that the apoptosis induced by ER stress is regarded as a precursor to the inflammatory reaction, and is a central mechanism, which leads to the development and progression of cardiovascular diseases, including atherosclerosis, ischemic heart diseases and heart failure (19-21), which all exhibit markedly increased FGF21 expression levels. However, whether the enhanced expression level of FGF21 is associated with ER stress in cardiomyocytes, and the functional role and molecular mechanism of FGF21 in ER stress-induced myocardial apoptosis remain unknown. Materials and methods Reagents. Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The rabbit anti β -Klotho (cat. no. AV53325) and mouse anti‑c‑myc (cat. no. M5546) antibodies were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The rabbit anti-FGF21 (cat. no. Ab171941), rabbit anti-phosphorylated (p)-FGFR1 (cat. no. Ab59194) and rabbit anti-p-IRE1α (cat. no. Ab124945) antibodies were obtained from Abcam (Cambridge, MA, USA). The rabbit anti-FGFR1 (cat. no. 9740), rabbit anti‑extracellular signal-regulated kinases (ERK)1/2 (cat. no. 4695), rabbit anti‑p‑ERK1/2 (cat. no. 4370), rabbit antiPERK (cat. no. 3192), rabbit anti-p-PERK (cat. no. 3179), rabbit anti‑ATF4 (cat. no. 11815), rabbit anti-IRE1α (cat. no. 3294), rabbit anti-JNK (cat. no. 9252), rabbit anti‑p‑JNK (cat. no. 9251), rabbit anti-eIF2α (cat. no. 5324), rabbit antip-eIF2α (cat. no. 3398) and rabbit anti-cleaved-caspase-3 (c-caspase-3; cat. no. 9661) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The rabbit anti-CHOP (cat. no. SC-7351) and mouse anti-β-actin
(cat. no. SC-47778) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The rabbit anti-Bcl-2 (cat. no. BA0315) and rabbit anti-Bcl-2 associated X protein (cat. no. BA0412) antibodies were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China). Antimouse IgG (cat. no. 7076) and anti-rabbit IgG (cat. no. 7074), PD166866 and PD98059 were purchased from Cell Signaling Technology. Tunicamycin (TM) was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Cell culture. Rat H9c2 cardiomyocytes (Chinese Academy of Sciences Cell Bank, Shanghai, China) were cultured in high glucose DMEM supplemented with 10% (v/v) FBS and 2 mM L-glutamine at 37˚C under a 5% CO2 atmosphere. For the present study H9c2 cells were plated at ~60% density and incubated overnight to reach 70-80% confluence at 37˚C before experimentation. Recombinant plasmid construction and transfection. The cDNA encoding full-length rat FGF21 (627 bp; accession no. NM_ 130752.1) was cloned and inserted into the XhoI and EcoRI restriction sites of pcDNA4/myc-his (5,151 bp) to generate the expression plasmid for rat FGF21 (pcDNA4FGF21). The pcDNA4-FGF21 plasmid was verified by special digestion analysis and DNA sequencing and transfected into cells using X-tremeGENE HP DNA Transfection reagent (Roche Diagnostics, Laval, Qc, Canada) for 48 h. Experimental protocols. The cultured H9c2 cardiomyocytes were randomly divided into different groups. In the control group, the H9c2 cardiomyocytes were incubated under normal conditions. In the TM-treated group, the H9c2 cardiomyocytes were treated with various doses (1, 5, 10, 20 and 50 µM) of TM for 24 h. In the FGF21 overexpressiontreated group (pcDNA4-FGF21 + TM), H9c2 cardiomyocytes were transfected with pcDNA4-FGF21 plasmid for 48 h and subsequently exposed to TM (10 µM) treatment for a further 24 h. Inhibitor-treated groups were processed the same as the pcDNA4-FGF21 + TM group, but the cells were co-incubated with the specific FGFR1 inhibitor, PD166866 (100 nM) or ERK1/2 inhibitor, PD98059 (20 µM) for 1 h before treatment with the pcDNA4-FGF21 plasmid. Three experimental categories were included: i) the control and TM (1, 5, 10, 20 and 50 µM) groups; ii) the control, TM (10 µM), pcDNA4-FGF21 + TM (10 µM), and pcDNA4 + TM (10 µM) groups; iii) the TM (10 µM), pcDNA4-FGF21 + TM (10 µM), pcDNA4-FGF21 + TM (10 µM) + PD166866, and pcDNA4FGF21 + TM (10 µM) + PD98059 groups. Cell viability assay. Cell viability was measured using Cell Counting Kit-8 (CCK-8, cat. no. CK04; Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's instructions. Cells were dispensed in 96-well plates at a density of 7x103 cells/well and incubated overnight at 4˚C. After the designated treatment, cells were washed in DMEM and 10 µl CCK-8 solution was added to each well for a 4-h incubation. Subsequently, the optical density (OD) of each well was measured at a wavelength of 450 nm using a spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). Cell viability (%) = (ODsample - ODblank)/(ODcontrol - ODblank) x 100%.
INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 40: 1477-1485, 2017
FGF21 secretion level measurement. H9c2 cells were maintained in medium containing 0.2% BSA rather than 10% FBS and treated with various concentrations of TM for 24 h. FGF21 released in the culture medium under ER stress conditions was determined using an ELISA kit (cat. no. MF2100; R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's instructions. All reactions were performed in triplicate using 96-well ELISA plates. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) staining. TUNEL assay was performed using a One Step TUNEL apoptosis kit (cat. no. KGA4052; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) according to the manufacturer's instruction. Cells were plated on glass coverslips at a density of 1x105 cells/well. Following treatment, the cells were fixed in 4% formaldehyde for 30 min and permeabilized in 0.5% Triton X-100 for 10 min. The cells were labeled with dUTP and TDT enzymes in a humidified box at 37˚C for 1 h, subsequently incubated in streptavidin-fluorescein (SF) at room temperature for 30 min and counterstained with 4',6-diamidino-2-phenylindole for 10 min before observation under a laser scanning confocal microscope (Bio-Rad Laboratories, Inc., Hercules, CA, USA). TUNEL-positive cells exhibited a bright green fluorescence and >5 non-overlapping fields in each group were quantified. Protein extraction and western blot analysis. Total protein was extracted according to the methods used by Kurian et al (22). Protein concentration was determined using a BCA protein assay kit (cat. no. P0012; Beyotime Institute of Biotechnology, Haimen, China) with BSA as standard. Total proteins (30 µg) were fractionated by 8-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon P; Millipore, Bedford, MA, USA) at 90 V constant for 100 min. The blots were blocked with 5% BSA for 1 h and the membranes were incubated with antibodies diluted in 5% BSA at 4˚C overnight. The primary antibodies used in the present study were as follows: FGF21 (1:500 dilution), FGFR1 (1:250 dilution), p-FGFR1 (1:200 dilution), β-Klotho (1:500 dilution), c-myc (1:1,000 dilution), ERK1/2 (1:3,000 dilution), p-ERK1/2 (1:3,000 dilution), PERK (1:1,000 dilution), p-PERK (1:1,000 dilution), eIF2α (1:750 dilution), p-eIF2α (1:1,000 dilution), ATF4 (1:5,000 dilution), CHOP (1:1,000 dilution), IRE1α (1:1,000 dilution), p-IRE1α (1:1,000 dilution), JNK(1:1,000 dilution), p-JNK (1:1,000 dilution), Bcl-2 (1:200 dilution), Bax (1:200 dilution), c-caspase-3 (1:200 dilution) and β-actin (1:2,000 dilution). After washing three times with 0.1% Tris-buffered saline with Tween-20 (TBST) and incubating with HRP-conjugated secondary antibodies [anti-mouse IgG (1:5,000 dilution) and anti-rabbit IgG (1:5,000 dilution)] for 1 h at room temperature, specific bands were visualized by enhanced chemiluminescence detection (cat. no. 34077; Thermo Fisher Scientific, Inc.). Bands were analyzed by the ImageJ software (National Institutes of Health, Bethesda, MD, USA). Each experiment was repeated at least three times. Statistical analysis. Statistical analysis was performed using the SPSS 19.0 software (SPSS, Inc., Chicago, IL, USA).
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Significant differences between the groups were analyzed by the unpaired Student's t-test or the one-way ANOVA and P10 µM and cell viability reduced with increased concentrations of TM. Thus, 10 µM TM was used in subsequent experiments for induction of ER stress. In addition, the PERK-eIF2α-AFT4-CHOP signaling pathway and IRE1α-JNK mediated signaling pathway are well known ER stress-associated signaling pathways for inducing apoptosis. Following treatment with TM up to 5 µM, mild ER stress partly activated these signaling pathways, which was evidenced by induction of p-PERK, p-eIF2α, ATF4 and CHOP in a dosedependent manner (Fig. 1B). In addition, mild ER stress caused upregulated expression of p-IRE1α, p-JNK, c-caspase-3 and Bax while it reduced the level of anti-apoptosis protein, Bcl-2 (Fig. 1C). However, at a high concentration (≥10 µM) of TM, this increasing level reached a maximum at a concentration of 10 µM TM and plateaued with the increasing concentration, this indicated that excessive ER stress (≥10 µM TM) fully activated ER stress-associated signaling pathways to induce further cell apoptosis. Mild ER stress elevates the expression levels of FGF21 and its main receptor in H9c2 cells. TM treatment significantly induced endogenous FGF21, p-FGFR1 and β-Klotho expression levels in H9c2 cells (Fig. 2A). Furthermore, increased levels of secreted FGF21 protein were detected from the culture medium of TM-treated H9c2 cells (Fig. 2B). However, the elevated expression level and secretion of these proteins were not positively correlated with the degree of ER stressinduced cell damage. Notably, in mild ER stress (≤5 µM TM), the protein expression level of FGF21, p-FGFR1 and β-Klotho increased and peaked at a concentration of 5 µM TM. However, in excessive ER stress (≥10 µM TM), the expression level of FGF21 and its main receptors decreased gradually with aggravation of the ER stress level. These results indicated that the elevated expression of FGF21 and its main receptors may be a compensatory response to injury in mild ER stress. FGF21 prevents the ER stress-induced apoptosis of H9c2 cells. FGF21 was overexpressed from the pcDNA4-FGF21 eukaryotic expression vector in H9c2 cells (Fig. 3A and B). To demonstrate the cardioprotective effect of FGF21 on ER stress-induced cardiomyocyte injury, H9c2 cells overexpressing FGF21 were exposed to TM (10 µM) treatment for 24 h. The result demonstrated that FGF21 overexpression significantly reversed the decreased cell viability caused by TM treatment, indicating that FGF21 protected H9c2 cells
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Figure 1. TM inhibited the cell viability of H9c2 cells and induced ER stress-associated signaling pathways. H9c2 cells were treated with various concentrations (1, 5, 10, 20 and 50 µM) of TM for 24 h. (A) Cell viability was detected by Cell Counting Kit-8 assay. (B) Western blot analysis of the expression levels of PERK-eIF2α-ATF4-CHOP signaling pathway-associated key proteins, including p-PERK, PERK, p-eIF2α, eIF2α, ATF4 and CHOP. (C) Western blot analysis of IRE1α-JNK signaling pathway-associated proteins, including p-IRE1α, IRE1α, p-JNK, JNK, c-caspase-3, Bax and Bcl-2. β-actin served as a loading control. Error bars demonstrate the means. *P
Fibroblast growth factor 21 protects rat cardiomyocytes from endoplasmic reticulum stress by promoting the fibroblast growth factor receptor 1-extracellular signal‑regulated kinase 1/2 signaling pathway Pingping Liang1*, Lin Zhong1*, Lei Gong2, Jiahui Wang3, Yujie Zhu1, Weifeng Liu1 and Jun Yang1 1
Department of Cardiology, 2Biochip Laboratory, and 3Central Laboratory, Yantai Yuhuangding Hospital, Affiliated Hospital of Medical College Qingdao University, Yantai, Shandong 264000, P.R. China Received February 25, 2017; Accepted September 8, 2017 DOI: 10.3892/ijmm.2017.3140
Abstract. Fibroblast growth factor 21 (FGF21), as an endocrine factor, is secreted into circulation by injured cardiomyocytes. Endoplasmic reticulum (ER) stress-induced apoptosis has been proposed as an important pathophysiological mechanism for cardiomyocyte injury. However, whether the enhanced expression of FGF21 in cardiomyocytes is linked to ER stress, and the effect and underlying mechanism of FGF21 on ER stress-induced cardiomyocyte apoptosis remain unclear. In the present study, it was demonstrated that mild ER stress resulted in upregulated expression levels of FGF21 and its main receptors, as a response to cell compensation, at the induction of ≤5 µM tunicamycin (TM). However, excessive ER stress (TM ≥10 µM) activated the ER stress-mediated apoptosis signaling pathways, including PKR-like ER kinase (PERK)-eukaryotic translational initiation factor 2α (eIF2α)-activating transcrip-
tion factor 4 (ATF4)-CCAAT/-enhancer-binding protein homologous protein (CHOP) and inositol-requiring kinase 1α (IRE1α)-c-Jun N-terminal kinases (JNK), as well as inhibited the expression of FGF21 and its primary receptors. In addition, FGF21 overexpression provided protection against ER stress-induced cardiomyocyte injury, as evidenced by increased cell viability and reduced apoptosis. These changes were associated with the inhibition of ER stress-mediated apoptosis signaling pathways, as well as increased phosphorylation of FGFR1 and ERK1/2. However, the protective effects of overexpressed FGF21 were abolished following treatment with FGFR1 and ERK1/2 inhibitors. Thus, mild ER stress may induce the expression of FGF21 and its primary receptors in cardiomyocytes. FGF21 inhibits ER stress-induced cardiomyocyte injury as least in part via the FGFR1-ERK1/2 signaling pathway. Introduction
Correspondence to: Dr Jun Yang, Department of Cardiology, Yantai Yuhuangding Hospital, Affiliated Hospital of Medical College Qingdao University, 20 Yuhuangding East Road, Yantai, Shandong 264000, P.R. China E-mail: [email protected] *
Contributed equally
Abbreviations: FGF21, fibroblast growth factor 21; ER, endo plasmic reticulum; UPR, unfolded protein response; PERK, PKR-like ER kinase; IRE1, inositol-requiring kinase 1α; ATF6, activating transcription factor 6; eIF2α, eukaryotic translational initiation factor 2α; ATF4, activating transcription factor 4; XBP1, X-box binding protein 1; JNK, c-Jun N-terminal kinase; CHOP, CCAAT/-enhancer-binding protein homologous protein; c-caspase-3, cleaved caspase-3; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2 associated X, apoptosis regulator; ERK, extracellular signalregulated kinases; TM, tunicamycin Key words: fibroblast growth factor 21, apoptosis, endoplasmic reticulum stress, cardiomyocytes, fibroblast growth factor receptor 1, extracellular signal-regulated kinase 1/2
Fibroblast growth factor 21 (FGF21) is a unique member of the FGF superfamily, which is predominantly expressed in the liver and exerts pleiotropic effects on metabolic processes (1). FGF21 may activate FGF receptors (FGFRs), including FGFR1-4, which all contain an intracellular tyrosine kinase domain, and FGFR1 is the predominant FGFR isotype in the heart and vessels (2). β-Klotho, as a co-receptor, is necessary for FGF21 to bind with particular FGFRs and effectively activate the FGF21 signaling pathway (3). Furthermore, among the various isoforms of FGFRs, FGFR1 has been suggested as the primary form for FGF21 in cardiomyocytes and the function of FGF21 was mediated primarily by binding to FGFR1 in a β -Klotho-dependent manner (2,4). A previous study identified that FGF21 is upregulated and released into circulation by the liver tissue in response to myocardial ischemia (5). Serum levels of FGF21 are also elevated in patients with coronary artery disease, acute myocardial infarction and heart failure (6-8). Additionally, FGF21 may decrease ischemia-reperfusion injury of cardiomyocytes and improve the antioxidant capacity of endothelial cells (9,10), indicating that FGF21 is a critical protective factor for the cardiovascular system.
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LIANG et al: FGF21 PROTECTS RAT CARDIOMYOCYTES FROM ENDOPLAMIC RETICULUM STRESS
Accumulation of unfolded or misfolded proteins at the reticulum transmembrane causes endoplasmic reticulum (ER) stress, activating an adaptive response termed the unfolded protein response (UPR) (11). The UPR involves the PKR-like ER kinase (PERK), inositol-requiring kinase 1α (IRE1α) and activating transcription factor 6 (ATF6) (12), which all regulate the transcription of a variety of associated genes encoding ER chaperone proteins to clear the unfolded protein aggregation at the initial stage of ER stress. When ER stress is prolonged and/or excessive, the UPR fails to control the level of unfolded or misfolded proteins in the ER, subsequently leading to the activation of apoptotic pathways (11-13). Proteins downstream of the PERK and IRE1 signaling pathways have been identified as having pro-apoptotic roles. PERK phosphorylates eIF2α to induce the translation of ATF4. ATF4 modulates a wide range of genes involved in the adaptation to ER stress and induces CCAAT/-enhancer-binding protein homologous protein (CHOP) to initiate the apoptosis signaling pathway (14,15). IRE1α mediates the accumulation of misfolded proteins, and induces downstream molecular X-box binding protein 1 (XBP1) and c-Jun N-terminal kinase (JNK). However, IRE1α-dependent apoptotic signaling occurs via phosphorylating JNK (16), which induces cell death via the induction of various proteins from the B-cell lymphoma-2 (Bcl-2) family and the activation of cysteinyl aspartate specific proteinase (caspases) (17,18). Evidence indicates that the apoptosis induced by ER stress is regarded as a precursor to the inflammatory reaction, and is a central mechanism, which leads to the development and progression of cardiovascular diseases, including atherosclerosis, ischemic heart diseases and heart failure (19-21), which all exhibit markedly increased FGF21 expression levels. However, whether the enhanced expression level of FGF21 is associated with ER stress in cardiomyocytes, and the functional role and molecular mechanism of FGF21 in ER stress-induced myocardial apoptosis remain unknown. Materials and methods Reagents. Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The rabbit anti β -Klotho (cat. no. AV53325) and mouse anti‑c‑myc (cat. no. M5546) antibodies were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The rabbit anti-FGF21 (cat. no. Ab171941), rabbit anti-phosphorylated (p)-FGFR1 (cat. no. Ab59194) and rabbit anti-p-IRE1α (cat. no. Ab124945) antibodies were obtained from Abcam (Cambridge, MA, USA). The rabbit anti-FGFR1 (cat. no. 9740), rabbit anti‑extracellular signal-regulated kinases (ERK)1/2 (cat. no. 4695), rabbit anti‑p‑ERK1/2 (cat. no. 4370), rabbit antiPERK (cat. no. 3192), rabbit anti-p-PERK (cat. no. 3179), rabbit anti‑ATF4 (cat. no. 11815), rabbit anti-IRE1α (cat. no. 3294), rabbit anti-JNK (cat. no. 9252), rabbit anti‑p‑JNK (cat. no. 9251), rabbit anti-eIF2α (cat. no. 5324), rabbit antip-eIF2α (cat. no. 3398) and rabbit anti-cleaved-caspase-3 (c-caspase-3; cat. no. 9661) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The rabbit anti-CHOP (cat. no. SC-7351) and mouse anti-β-actin
(cat. no. SC-47778) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The rabbit anti-Bcl-2 (cat. no. BA0315) and rabbit anti-Bcl-2 associated X protein (cat. no. BA0412) antibodies were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China). Antimouse IgG (cat. no. 7076) and anti-rabbit IgG (cat. no. 7074), PD166866 and PD98059 were purchased from Cell Signaling Technology. Tunicamycin (TM) was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Cell culture. Rat H9c2 cardiomyocytes (Chinese Academy of Sciences Cell Bank, Shanghai, China) were cultured in high glucose DMEM supplemented with 10% (v/v) FBS and 2 mM L-glutamine at 37˚C under a 5% CO2 atmosphere. For the present study H9c2 cells were plated at ~60% density and incubated overnight to reach 70-80% confluence at 37˚C before experimentation. Recombinant plasmid construction and transfection. The cDNA encoding full-length rat FGF21 (627 bp; accession no. NM_ 130752.1) was cloned and inserted into the XhoI and EcoRI restriction sites of pcDNA4/myc-his (5,151 bp) to generate the expression plasmid for rat FGF21 (pcDNA4FGF21). The pcDNA4-FGF21 plasmid was verified by special digestion analysis and DNA sequencing and transfected into cells using X-tremeGENE HP DNA Transfection reagent (Roche Diagnostics, Laval, Qc, Canada) for 48 h. Experimental protocols. The cultured H9c2 cardiomyocytes were randomly divided into different groups. In the control group, the H9c2 cardiomyocytes were incubated under normal conditions. In the TM-treated group, the H9c2 cardiomyocytes were treated with various doses (1, 5, 10, 20 and 50 µM) of TM for 24 h. In the FGF21 overexpressiontreated group (pcDNA4-FGF21 + TM), H9c2 cardiomyocytes were transfected with pcDNA4-FGF21 plasmid for 48 h and subsequently exposed to TM (10 µM) treatment for a further 24 h. Inhibitor-treated groups were processed the same as the pcDNA4-FGF21 + TM group, but the cells were co-incubated with the specific FGFR1 inhibitor, PD166866 (100 nM) or ERK1/2 inhibitor, PD98059 (20 µM) for 1 h before treatment with the pcDNA4-FGF21 plasmid. Three experimental categories were included: i) the control and TM (1, 5, 10, 20 and 50 µM) groups; ii) the control, TM (10 µM), pcDNA4-FGF21 + TM (10 µM), and pcDNA4 + TM (10 µM) groups; iii) the TM (10 µM), pcDNA4-FGF21 + TM (10 µM), pcDNA4-FGF21 + TM (10 µM) + PD166866, and pcDNA4FGF21 + TM (10 µM) + PD98059 groups. Cell viability assay. Cell viability was measured using Cell Counting Kit-8 (CCK-8, cat. no. CK04; Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's instructions. Cells were dispensed in 96-well plates at a density of 7x103 cells/well and incubated overnight at 4˚C. After the designated treatment, cells were washed in DMEM and 10 µl CCK-8 solution was added to each well for a 4-h incubation. Subsequently, the optical density (OD) of each well was measured at a wavelength of 450 nm using a spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). Cell viability (%) = (ODsample - ODblank)/(ODcontrol - ODblank) x 100%.
INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 40: 1477-1485, 2017
FGF21 secretion level measurement. H9c2 cells were maintained in medium containing 0.2% BSA rather than 10% FBS and treated with various concentrations of TM for 24 h. FGF21 released in the culture medium under ER stress conditions was determined using an ELISA kit (cat. no. MF2100; R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's instructions. All reactions were performed in triplicate using 96-well ELISA plates. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) staining. TUNEL assay was performed using a One Step TUNEL apoptosis kit (cat. no. KGA4052; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) according to the manufacturer's instruction. Cells were plated on glass coverslips at a density of 1x105 cells/well. Following treatment, the cells were fixed in 4% formaldehyde for 30 min and permeabilized in 0.5% Triton X-100 for 10 min. The cells were labeled with dUTP and TDT enzymes in a humidified box at 37˚C for 1 h, subsequently incubated in streptavidin-fluorescein (SF) at room temperature for 30 min and counterstained with 4',6-diamidino-2-phenylindole for 10 min before observation under a laser scanning confocal microscope (Bio-Rad Laboratories, Inc., Hercules, CA, USA). TUNEL-positive cells exhibited a bright green fluorescence and >5 non-overlapping fields in each group were quantified. Protein extraction and western blot analysis. Total protein was extracted according to the methods used by Kurian et al (22). Protein concentration was determined using a BCA protein assay kit (cat. no. P0012; Beyotime Institute of Biotechnology, Haimen, China) with BSA as standard. Total proteins (30 µg) were fractionated by 8-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon P; Millipore, Bedford, MA, USA) at 90 V constant for 100 min. The blots were blocked with 5% BSA for 1 h and the membranes were incubated with antibodies diluted in 5% BSA at 4˚C overnight. The primary antibodies used in the present study were as follows: FGF21 (1:500 dilution), FGFR1 (1:250 dilution), p-FGFR1 (1:200 dilution), β-Klotho (1:500 dilution), c-myc (1:1,000 dilution), ERK1/2 (1:3,000 dilution), p-ERK1/2 (1:3,000 dilution), PERK (1:1,000 dilution), p-PERK (1:1,000 dilution), eIF2α (1:750 dilution), p-eIF2α (1:1,000 dilution), ATF4 (1:5,000 dilution), CHOP (1:1,000 dilution), IRE1α (1:1,000 dilution), p-IRE1α (1:1,000 dilution), JNK(1:1,000 dilution), p-JNK (1:1,000 dilution), Bcl-2 (1:200 dilution), Bax (1:200 dilution), c-caspase-3 (1:200 dilution) and β-actin (1:2,000 dilution). After washing three times with 0.1% Tris-buffered saline with Tween-20 (TBST) and incubating with HRP-conjugated secondary antibodies [anti-mouse IgG (1:5,000 dilution) and anti-rabbit IgG (1:5,000 dilution)] for 1 h at room temperature, specific bands were visualized by enhanced chemiluminescence detection (cat. no. 34077; Thermo Fisher Scientific, Inc.). Bands were analyzed by the ImageJ software (National Institutes of Health, Bethesda, MD, USA). Each experiment was repeated at least three times. Statistical analysis. Statistical analysis was performed using the SPSS 19.0 software (SPSS, Inc., Chicago, IL, USA).
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Significant differences between the groups were analyzed by the unpaired Student's t-test or the one-way ANOVA and P10 µM and cell viability reduced with increased concentrations of TM. Thus, 10 µM TM was used in subsequent experiments for induction of ER stress. In addition, the PERK-eIF2α-AFT4-CHOP signaling pathway and IRE1α-JNK mediated signaling pathway are well known ER stress-associated signaling pathways for inducing apoptosis. Following treatment with TM up to 5 µM, mild ER stress partly activated these signaling pathways, which was evidenced by induction of p-PERK, p-eIF2α, ATF4 and CHOP in a dosedependent manner (Fig. 1B). In addition, mild ER stress caused upregulated expression of p-IRE1α, p-JNK, c-caspase-3 and Bax while it reduced the level of anti-apoptosis protein, Bcl-2 (Fig. 1C). However, at a high concentration (≥10 µM) of TM, this increasing level reached a maximum at a concentration of 10 µM TM and plateaued with the increasing concentration, this indicated that excessive ER stress (≥10 µM TM) fully activated ER stress-associated signaling pathways to induce further cell apoptosis. Mild ER stress elevates the expression levels of FGF21 and its main receptor in H9c2 cells. TM treatment significantly induced endogenous FGF21, p-FGFR1 and β-Klotho expression levels in H9c2 cells (Fig. 2A). Furthermore, increased levels of secreted FGF21 protein were detected from the culture medium of TM-treated H9c2 cells (Fig. 2B). However, the elevated expression level and secretion of these proteins were not positively correlated with the degree of ER stressinduced cell damage. Notably, in mild ER stress (≤5 µM TM), the protein expression level of FGF21, p-FGFR1 and β-Klotho increased and peaked at a concentration of 5 µM TM. However, in excessive ER stress (≥10 µM TM), the expression level of FGF21 and its main receptors decreased gradually with aggravation of the ER stress level. These results indicated that the elevated expression of FGF21 and its main receptors may be a compensatory response to injury in mild ER stress. FGF21 prevents the ER stress-induced apoptosis of H9c2 cells. FGF21 was overexpressed from the pcDNA4-FGF21 eukaryotic expression vector in H9c2 cells (Fig. 3A and B). To demonstrate the cardioprotective effect of FGF21 on ER stress-induced cardiomyocyte injury, H9c2 cells overexpressing FGF21 were exposed to TM (10 µM) treatment for 24 h. The result demonstrated that FGF21 overexpression significantly reversed the decreased cell viability caused by TM treatment, indicating that FGF21 protected H9c2 cells
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LIANG et al: FGF21 PROTECTS RAT CARDIOMYOCYTES FROM ENDOPLAMIC RETICULUM STRESS
Figure 1. TM inhibited the cell viability of H9c2 cells and induced ER stress-associated signaling pathways. H9c2 cells were treated with various concentrations (1, 5, 10, 20 and 50 µM) of TM for 24 h. (A) Cell viability was detected by Cell Counting Kit-8 assay. (B) Western blot analysis of the expression levels of PERK-eIF2α-ATF4-CHOP signaling pathway-associated key proteins, including p-PERK, PERK, p-eIF2α, eIF2α, ATF4 and CHOP. (C) Western blot analysis of IRE1α-JNK signaling pathway-associated proteins, including p-IRE1α, IRE1α, p-JNK, JNK, c-caspase-3, Bax and Bcl-2. β-actin served as a loading control. Error bars demonstrate the means. *P
